Phosphatidyl oligoglycerols

ABSTRACT

In order to form liposomes with a longer half-life in blood, use is made of defined formula (A)

[0001] The invention relates to phosphatidyl compounds which contain adefined hydrophilic residue, and to long-circulating liposomes.

[0002] Conventional liposomes circulate in the serum for up to 5 hours.However, especially when liposomes are used as a means of drug delivery,it is desirable that they circulate in the bloodstream for as long aspossible.

[0003] To this end, the so-called “stealth liposomes” were developed,which are not destroyed in the bloodstream so quickly. These “stealthliposomes” are built up on the basis of phosphatidyl compounds whichhave an extended polyethylene glycol residue. The polyethylene glycolresidue proved to be most effective in producing the desired increase inliposome survival duration when the molecular weight was between 2000and 3000. A serious disadvantage, however, of these “stealth-liposomes”,ie, of these phosphatidyl compounds with a polyethylene glycol residue,is that the compounds are not exactly defined, since the polyethyleneglycol residues display different chain lengths.

[0004] Maruyama et al. (Int. J. Pharmac. 111 (1994), 103-107) suggestedthe use of dipalmitoyl phosphatidyl polyglycerols to lengthen theduration of liposome circulation. However, since technical-gradepolyglycerols were used as starting material, no uniform products wereobtained here either. Technical polyglycerols, which consist of amixture of polyglycerols with different chain lengths and monoglycerol,and which are characterized by their average molecular weight, werephosphatidylated by means of phospholipase D. The resulting productsonly led to a small increase in the survival duration of liposomes inthe blood.

[0005] The object of this invention was thus to provide compounds whichincrease the survival duration of liposomes and which are of exactlydefinable composition.

[0006] This objective is established according to the invention by meansof a compound with the general formula (A)

[0007] where R¹ and R², independent of each other, stand for hydrogen ora saturated or unsaturated alkyl or acyl residue, which may be branchedand/or substituted, R³ stands for hydrogen or an alkyl residue,

[0008] n=0 or 1,

[0009] x is a whole number from 1 to 4 and

[0010] m is a whole number from 2 to 10 if n=0, or a whole number from 1to 10 if n=1, or 1 if x is greater than 1,

[0011] and where, in the case that n=0, the compound is more than 90%uniform with respect to the value of m.

[0012] The stepwise synthesis—upon which this invention is based—of thehydrophilic residues of the phosphatidyl compounds of formula (A) makesit possible to obtain compounds of an exactly defined composition.

[0013] The compound of the invention, with the formula (A), is thus nota mixture of various molecules of indefinite composition and chainlength, but can be synthesized to have precisely the desired structure.If, for example, the desired product is a triglycerol derivative, ie,x=1 arid m=3 in formula (A), the content of monoglycerol, diglycerol,tetraglycerol and higher oligoglycerol derivatives will be low. It ispreferable if a glycerol derivative of a certain chain length isobtained that is largely free of glycerol derivatives of other chainlengths. The content of monoglycerol derivatives, in particular, is low,amounting to less than 5%, preferably less than 1% or, even morepreferably, less than 0.1% relative to the desired oligoglycerolderivative.

[0014] According to the invention, the compound of formula (A) is auniform compound of defined structure. It is of advantage if, withrespect to the value of m, the compound is greater than 95% uniform. Itis to greater advantage if it is more than 99% uniform. It is evenpossible to provide a compound which is more than 99.9% uniform withrespect to the value of m.

[0015] The compound is preferably an oligoglycerol derivative with 2 to5 glycerol units, more preferably with 2 to 4 glycerol units. It is toadvantage if these are 1.3-linked linear oligoglycerol residues.

[0016] According to the invention, the residues R¹ and R², independentof each other, stand for hydrogen, a saturated or unsaturated C₁-C₂₄alkyl or C₁-C₂₄ acyl residue, preferably hydrogen or a saturated orunsaturated C₈-C₂₄ alkyl or C₈-C₂₄ acyl residue, it being beneficial ifit at least one of the residues R¹ and R² is an acyl residue.

[0017] The residue R³ is preferably hydrogen or an alkyl residue with 1to 4 carbon atoms.

[0018] The compound of formula (A) can be a racemic compound whichcontains a phospho-rac-(1 or 3)-oligoglycerol linkage, or it can be inthe form of a stereospecific isomer. The stereoisomers can have aphospho-sn-1-oligoglycerol linkage or a phospho-sn-3-oligoglycerollinkage. The formation of the stereospecific linkage can be carried outin a manner analogous to those described in the literature (DE 31 30 867A1; H. Eibl et al., Chem. Phys. Lipids, 28 (1981), 1-5, 41 (1986), 53-63and 47 (1988, 47-53).

[0019] The subject matter of the invention also includes liposomes whichcontain phospholipids and/or alkyl phospholipids, maybe cholesterol, and1 to 50 mol % of a compound with the general formula (A),

[0020] or salts thereof, where the cholesterol, the phospholipids, thealkyl phospholipids and the compound of formula (A) together make up 100mol %, and R¹ and R², independent of each other, stand for hydrogen, asaturated or unsaturated alkyl or acyl residue which may be branchedand/or substituted,

[0021] R³ stands for hydrogen or an alkyl residue,

[0022] n=0 or 1,

[0023] x is a whole number from 1 to 4 and

[0024] m is a whole number from 2 to 10 if n=0, or a whole number from 1to 10 if n=1, or 1 if x is greater than 1, and where, in the case thatn=0, the compound (A) is more than 90% uniform in respect of the valueof m.

[0025] The liposomes of the invention have a half-life in serum of up to18 to 20 hours. Surprisingly, the liposome concentration in blood wasfound to decrease linearly.

[0026] It is beneficial according to the invention if compound (A)displays a uniformity of more than 95% or, even better, more than 99%with respect to the value of m. It is also possible, however, to usecompound (A) in practically pure form, ie, more than 99.9% uniform withrespect to the value of m.

[0027] The liposomes preferably contain a compound of formula (A), inwhich x=1 and m is a whole number from 2 to 5; it is even morepreferable if m is a whole number from 2 to 4.

[0028] The residues R¹ and R² of the compound of formula (A) containedin the liposomes can stand, independent of each other, for hydrogen or asaturated or unsaturated C₁-C₂₄ alkyl or C₁-C₂₄ acyl residue, preferablyhydrogen or a saturated or unsaturated C₈-C₂₄ alkyl or C₈-C₂₄ acylresidue. The substituent is a residue which does not interfere duringthe preparation. R³ is preferably hydrogen or a C₁-C₄ alkyl residue.

[0029] The compound of formula (A) can be present in the liposomes as aracemic mixture, ie, with a phospho-rac-(1 or 3)-oligoglycerol linkage.It is preferable if it is present in stereospecific form with aphospho-sn-1-oligoglycerol linkage or a phospho-sn-3-oligoglycerollinkage.

[0030] It is to advantage if at least one of the residues R¹ and R² offormula (A) is an acyl group.

[0031] It is beneficial if liposomes containing the compound of formula(A) with n=0 exhibit an excess negative charge. However, liposomes canalso be prepared from compounds of formula (A) in which n=1. In thiscase, it is better if the liposomes exhibit no excess charge or apositive one.

[0032] Besides a compound of formula (A), the liposomes containphospholipids and/or alkyl phospholipids and maybe cholesterol. It ispreferable to use the compound of formula (A) in an amount of 5 to 15mol %. If the liposomes do not display an excess charge, a compositionof 0 to 70 mol % cholesterol, 1 to 50 mol % of a compound of formula(A), and phospholipids and/or alkyl phospholipids is preferred. If thereis a negative excess charge, a preferred liposome composition consistsof 0 to 70 mol % cholesterol, 1 to 15 mol % of a compound of formula(A), and phospholipids and/or alkyl phospholipids. A higher proportionof compounds of formula (A) with a negative excess charge would lead toinstability of the liposomes in the blood circulation. It is toadvantage if the liposomes comprise 35 to 43 mol %, in particular 38 to42 mol % cholesterol, 5 to 15 mol % of a compound of formula (A), andphospholipids and/or alkyl phospholipids.

[0033] The phospholipids and/or alkyl phospholipids can, for example, bediacyl phospho-glycerols of defined structure. Generally speaking, theselipid components can be used as compounds of defined structure.

[0034] In the case that x>1, it is preferable if the residue—CH₂(—CHOH)_(x)—CH₂OH derives from sugar alcohols which have fourhydroxyl groups for x=2, five hydroxyl groups for x=3, and 6 hydroxylgroups for x=4. Examples of such residues are mannitol derivatives forx=4, lyxitol derivatives for x=3 and threitol derivatives for x=2.

[0035] The liposomes of the invention have a markedly longer half-lifein the blood stream. Their half-life is preferably at least 10 hours,better still, more than 12 hours. Half-lives of 18 to 20 hours have beenmeasured for the liposomes of the invention. Surprisingly, the decreasein blood lipid concentration with time was found to be absolutelylinear. It is preferable according to the invention if, after 6 hours,more than 50% of the liposomes added are still present in the blood; itis even more preferable if more than 60% are still present.

[0036] A particularly surprising property of the liposomes of theinvention is their preferred tendency to accumulate in the spleen.Depending on the composition and size of the liposomes, enrichmentthereof in the spleen has been found which exceeds enrichment in theliver by a factor of 25. Enrichment in the spleen compared with that inthe liver increases with increasing value of m in formula A and withincreasing size of the liposomes. With the transition from SUVs (SmallUnilamellar Liposomes; diameter about 60 nm) to LUVs (Large UnilamellarLiposomes; diameter about 190 nm), the degree of enrichment in thespleen increases many times over. The preferential accumulation in thespleen also increases as the number of carbon atoms in R¹ and R²increases.

[0037] It was found, in addition, that the liposomes of the inventionalso accumulate in certain tumour tissues. This was observed to be thecase, for example, with breast carcinomas induced by nitrosomethylurea(MNU carcinoma).

[0038] The liposomes of the invention can also contain one or morepharmaceutical drugs.

[0039] Generally speaking, all drugs can be used that can be introducedinto the plasma by means of liposomes. Preferred groups of drugs are, onthe one hand, cytostatic agents, especially anthracycline antibioticssuch as doxorubicin, epirubicin and daunomycin, with doxorubicip beingespecially preferred. Other preferred anti-tumour drugs are idanibicin,hexadecylphosphocholine,1-octadecyl-2-methyl-rac-glycero-3-phosphocholine, 5-fluoruracil,cis-platinum complexes such as carboplatin and novantron, andmitomycins.

[0040] Other preferred groups of drugs are immunomodulating substancessuch as citokines, of which interferon and, in particular, α-interferonare given special preference, antimycotic substances (eg, amphotericinB), and drugs to combat protozoan diseases (malaria and trypanosome andleishmania infections). Taxol is another preferred drug.

[0041] Yet another group of preferred drugs is the group of lytic drugs,as are described in the DE 41 32 345 A1. The content of this patentapplication is thus included by way of reference. Preferred drugs aremiltefosin, edelfosin, ilmofosin and SRI62-834.

[0042] The subject matter of the invention thus includes use of theliposomes according to the invention for preparing an anti-tumour agent,with the drug doxorubicin being given special preference.

[0043] The subject matter of the invention also includes use of theliposomes according to the invention for preparing an agent to influencecell proliferation, with the drug preferably being a cytokine, inparticular α-interferon.

[0044] The subject matter of the invention includes, in addition, apharmaceutical composition which contains the liposomes described aboveand, entrapped in the liposomes, one or more pharmaceutical drugs,combined if necessary with standard pharmaceutical diluents, adjuvants,carrier media and fillers.

[0045] The liposomes of the invention are prepared using methods whichare known per se and with the usual equipment. Typically, a solutioncontaining the various components of the liposome and 1 to 50 mol % of acompound of formula (A) is converted into a lipid suspension which isthen pressed under high pressure through nozzles or a perforated disk;the size of the liposomes can be regulated by means of the size of theperforations in the disk. Suitable measures for converting a lipidsuspension into liposomes are familiar to persons versed in the art.Preferably, 5 to 15 mol % of a compound of the general formula (A), 35to 43 mol % cholesterol and 42 to 60 mol % phospholipids and/or alkylphospholipids are converted into a lipid suspension, which in turn isconverted into liposomes by means of suitable measures and in a mannerknown per se.

[0046] These known methods can also be used to make a pharmaceuticalformulation which contains the liposomes of the invention and one ormore pharmaceutical drugs. To entrap water-insoluble drugs, the drug isdissolved together with the lipid components, while to entrapwater-soluble drugs, an aqueous solution which contains thewater-soluble drug is added to the lipid film.

[0047] The compounds of the invention, having the formula (A), can beprepared in cases where n=1 by linking a defined oligoglycerol with aphosphatidyl ethanolamine by way of the amino group. This results inneutral compounds, ie, compounds without an excess charge. The definedoligoglycerols used for linking are compounds with the formula (B).

[0048] In cases where n=0, compounds with the general formula (A) aremade by linking a defined oligoglycerol with a phosphatidylglycerol.When n=0, compounds with the general formula (A) can also be made—usinga phosphorylation agent—by linking a defined oligogycerol or a C₄-C₆sugar alcohol with an alcohol of the formula CH₂—OR¹—CHOR²—CHOH. Asphosphorylation agent, use is made preferably of POCl₃.

[0049] The preparation of phospholipids from diacyl glycerols isdescribed in the literature (DE 32 39 817 A1; P. Woolley et al., Chem.Phys. Lipids 47 (1988), 5562; H. Eibl et al., Chem. Phys. Lipids 47(1988), 63-68), and this method can be applied here.

[0050] Using the above-described methods, a racemic compound is formedwhich contains a phospho-rac-(1 or 3)-oligoglycerol linkage. It is toadvantage if stereospecific compounds are formed, which exhibit aphospho-sn-1-oligoglycerol linkage or a phospho-sn-3-oligoglycerollinkage. To make a compound of formula (A), it is preferable to use alinear oligoglycerol of defined chain length.

[0051] The subject matter of the invention also includes a protectedoligoglycerol of the formula (B),

[0052] where Y is a whole number from 1 to 9 and X is a benzyl, alkyl ortetrahydropropanyl group. It is beneficial if Y is a whole number from 1to 3. It is possible according to the invention to obtain 1.3-linkedoligoglycerols in practically pure form. Oligoglycerols of a predefinedchain length can be prepared which contain hardly any impurities in theform of oligoglycerols with different chain lengths. In addition, theseoligoglyerols of the invention are practically free of monomericglycerol. In other words, uniform compounds are obtained, which have adefined structure.

[0053] In the oligoglycerol, X can also stand for a different suitableprotective group. It is also possible to replace the acetone withanother protective group, in particular another ketone.

[0054] The invention comprises, in addition, alkyl oligoglycerols offormula (C)

[0055] where Y is a whole number from 0 to 8, preferably a whole numberfrom 1 to 3, and one of the residues X or Z is a saturated orunsaturated alkyl residue and the other residue is hydrogen. These alkyloligoglycerols are also uniform compounds of defined structure.

[0056] The production of oligoglycerols, protected oligoglycerols andalkyl oligoglycerols is of particular interest, because with the help ofthese starting materials a number of important and novel adjuvantsserving as solubilizers and to improve membrane permeation are obtained.Of particular interest with respect to increasing the period for whichthe liposomes survive in the blood stream is the production ofphosphatidyl oligoglycerol derivatives of formula (A), which carryadditional hydroxyl groups in the polar area.

[0057] Due to the preferred enrichment of the liposomes of the inventionin the spleen, these liposomes are suitable generally for the selectiveintroduction of substances into the spleen. These substances may bemedicinal products, contrast agents or the like. This is especiallyimportant with regard to improving the quality of vaccines, since thespleen plays a major role in the formation of antibodies for the immunesystem. In the same way, the enrichment of the liposomes according tothe invention such as was observed in tumour tissue is of importancewith regard to delivering drugs, contrast agents and the likespecifically to such tissue.

[0058] The following examples, together with the enclosed drawings,explain the invention in more detail. In the drawings:

[0059]FIG. 1 shows the total-organ distribution of liposomes accordingto the invention in the spleen and in the liver.

[0060]FIG. 2 shows the per-gram distribution of liposomes according tothe invention in the spleen and in the liver.

[0061]FIG. 3 shows how the blood levels of different liposomes of theinvention vary as a function of time.

EXAMPLE 1

[0062] In an animal experiment, liposomes were used which consisted of40 mol % cholesterol, 10 mol % phosphatidylglycerol and 50% dipalmitoyllecithin. The liposomes had a half-life in serum of 4 hours, with atypical persistence characteristic, ie, a rapid decrease to start with,followed by a slower decrease.

[0063] Liposomes of the same composition were prepared, in which thephosphatidylglycerol was replaced by a phosphatidylglycerol G2 of theinvention. A half-life in serum of 18 to 20 hours was measured, thedecrease with time being absolutely linear. This linear relation wasobserved irrespective of the size of the liposomes. The same linearreduction in serum liposome concentration was found with 50 nm liposomesand with 150 nm liposomes. The linear reduction in blood liposomeconcentration was also ob served for different starting concentrations.

EXAMPLE 2

[0064] Percentage of Liposomes in the Blood Stream After 6 Hours

[0065] Liposomes according to the invention were prepared, consisting ofdipalmitoyl-sn-G-3-PC/cholesterol/dipalmitoyl-sn-G-3-PGγ in a molarratio of 45:45:10. The percentages of liposomes still in the blood after6 hours are listed in Table 1. For comparison, the percentages measuredby Maruyama et al. under the same conditions for the systemdistearyl-sn-G-3-PC/cholesterol/dipalmitoyl-sn-G-3-PG_(Yi) 45:45:10 arelisted too. Compared to the prior art, the example of the inventionshows a pronounced increase in the quantity of liposomes found. TABLE 1Y Comparative example Y Example of the invention 0 18% 0 21% 2 19% 2 80%3 — 3 82% 4 20% 4 56%

EXAMPLE 3

[0066] Liposomes consisting of1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphoglyceroglycerol (PG_(n)) andcholesterol in a molar ratio of 4:1:5 were doped with tritium-labelledinulin. These liposomes were administered to rats in a dosage of 100mmol lipid per kg rat, and after 72 hours the distribution of theseliposomes in the spleen and the liver was determined by measuring theradioactivity. Liver weights varied between 9 and 10 g, those of thespleen between 0.6 and 0.7 g. FIG. 1 of the endosed drawings shows thatfor a liver weight which is about 15 times higher than that of thespleen, the distribution of liposomes (SUVs) increases substantially infavour of the spleen as the number of glycerol units increases (x=1; m=1to 4 in formula A).

[0067] In FIG. 2, the liposome uptake by the spleen and the liver isdepicted as uptake per gram of the organ. For n=4, the spleen is seen tohave a liposome concentration which is about 9 times higher than that ofthe liver; for n=1, the enrichment factor equals 4. In the last column,FIGS. 1 and 2 show the effect which the size of the liposomes has. ForLUVs with a diameter of 190 nm, liposome enrichment is even more infavour of the spleen: even when n only equals 2, the enrichment factorequals 24. In practical terms, this means that it is no longer possibleto target the liver with these liposomes.

EXAMPLE 4

[0068] Preparation of Compounds with the Formula (A)

EXAMPLE 4a Key Intermediate with the Formula I

[0069] The oligoglycerols diglycerol (G₂), triglycerol (G₃) andtetraglycerol (G₄) can be prepared from an easily obtained keyintermediate with the formula I,1.2-isopropylidene-rac-glycero-3.1-rac-glycero-3-allylether, (see modelA).

[0070] Model A: Oligoglycerols from Formula I

[0071] The intermediate product described by formula I can be obtainedin large quantities from commercial allyl glydidyl ether byNaOH-catalyzed ring opening with 1.2-isopropylidene-rac-glycerol, whichis likewise available in the chemicals trade:

Epoxide Opening with Alcohols (General Example)

[0072] Production of the key intermediate with the formula I:

[0073] 1.2-isopropylidene-rac-G₁-3.1-0.0-3-O-allyl-rac-G₂

[0074] A catalytic quantity of NaOH (MW 40.00; 0.6 mol-24 g) is added to1.2-isopropylidene-rac-glycerol (MW 132.16; 16 mol-2115 g), which isrendered a solution by stirring and heating to 80° C. At 80° C., allylglycidyl ether (MW 114.14; 6 mol-685 g) is added dropwise over a periodof 2 hours, and the reaction mixture stirred for another 2 hours at 80°C. By this point in time the epoxide (R_(f) in ether=0.8) has reactedcompletely to form the G₃ constituent (R_(f) in ether=0.6) The excessisopropylidene-rac-glycerol has an R_(f) of 0.65 in ether and is removedfrom the reaction mixture at 75° C./10 mbar. The residue has 1 ldiisopropyl ether added to it and is extracted twice with 1 l NaCl (1%solution in H₂O) in each case. The organic phase is rotated in anevaporator and distilled (Kpi₁₀-1 mbar 125° C.).

[0075] The yield of the pure product1.2-isopropylidene-rac-G₁-3.10.0-3-O-allyl-rac-G₂ (MW 246;30) is 1025 g(ca. 70%).

[0076] Instead of 1.2-isopropylidene-rac-glycerol, it is possible toreact other primary alcohols and also allyl alcohol and benzyl alcoholunder the given conditions. In the same way, it is also possible to useother epoxides.

[0077] The intermediate product with the formula I can also be obtainedfrom 1.2-isopropylidene-rac-glycero-3-glycidyl ether by means ofNaOH-catalyzed ring opening with allyl alcohol. In this case,1.2-isopropylidene-rac-glycero-3 glycidyl ether must first be made fromallyl glycerol.

EXAMPLE 4b:

[0078] Alkylation of Primary or Secondary Hydroxyl Groups (GeneralExample)

[0079] Preparation of a key intermediate:

[0080] 1.2-isopropylidene-rac-G₁-3.1-0.0-2-O-benzyl-3-O-allyl-rac-G₂

[0081] The key intermediate,1.2-isopropylidene-rac-G₁-3.1-rac-G₂-0-allyl ether (MW 246.30; 0.5mol-123 g) is dissolved in 500 ml tetrahydrofuran, has benzyl chloride(0.6 mol-76 g) added to it and is reflux-boiled. K-tert. butylate (0.7mol-79 g) dissolved in 500 ml tetrahydrofuran is added dropwise. Thereaction is completed after 30 minutes of reflux-boiling (TLCcheck—R_(f) in ethyl ether educt, R_(f)=0.1; product, R_(f)=0.4). Thereaction mixture has 1 l diisopropyl ether and 1 l 1% NaCl solutionadded to it, is shaken, and the upper phase rotated in an evaporator.The product can either be used directly, or recovered in pure form inapproximately 90% yield by means of chromatography on silica gel.

[0082] Empirical formula C₁₉H₂₈O₅ (MW 336.42) calculated: C, 67.83; H,8.39; 0, 23.79 measured: C, 67.78; H, 8.34; O, -

[0083] Instead of benzyl chloride, use can also be made of benzylbromide, allyl chloride or allyl bromide, or of the halogenides ormesylates of primary alcohols. The products of the reaction betweenprimary or secondary hydroxyl groups and alkyl mesylates, in particular,lead to high yields (>90%) of the desired target compounds.

EXAMPLE 4c:

[0084] Synthesis Sequence 0-Allyl Ether→0-Propenyl Ether→Alcohol(General Example)

[0085] Preparation of2-0-benzyl-rac-G₁-1.3-0.0-1.2-isopropylidene-rac-G₂

[0086] Rearrangement

[0087] 1.2-isopropylidene-rac-G₁-3.1-0.0-2-0-benzyl-3-0-allyl-rac-G₂(0.5 mol-168 g) is dissolved in 500 ml DMF, to which k-tert. butylate(0.7 mol-79 g) is then added. The reaction mixture is heated to 110 to115° C. with continuous stirring, left for 15 minutes at thistemperature and then cooled to 20° C. Following the addition of 500 mldiisopropyl ether and 500 ml 1% NaCl, the upper, diisopropyl ether phaseis removed and the solvent eliminated under vacuum (TLC check—Rf inhexane/diisopropyl ether (1:1): educt, R_(f)=0.2; product, R_(f)=0.4).

[0088] Cleavage of the Propenyl Protective Group

[0089] The residue from the above reaction, approximately 168 g, isdissolved in 500 ml methanol and, following addition of 50 ml 1 M HCl,is reflux-boiled. The reaction is complete after 60 minutes (TLC checkin hexane/diisopropyl ether (1:1): educt, R_(f)=0.4; product, R_(f)=0).The yield of rac-G₁-3.1-rac-G2-2-0-benzyl ether is >90%. Under theacidic conditions prevailing during propenyl cleavage, theisopropylidene protective group is likewise removed. If necessary, itcan be reintroduced in the 1.2 position.

[0090] Introduction of the Isopropylidene Protective Group

[0091] The residue from the above reaction (approx. 0.5 mol) isdissolved in 300 ml THF to which, in succession, 2.2-dimethoxypropane(0.5 mol-52 g) and 0.2 g H₂SO₄ in 10 ml THF are added, and then stirredfor 2 hours at 25° C. The reaction mixture is neutralized with saturatedNa₂CO₃ solution, the precipitate removed under suction and the filtraterotated with xylol under vacuum to free it of water. The product ispurified chromatographically on silica gel 60 (Merck, grain size 0.2-0.5mm) (R_(f) in diethyl ether: educt, R_(f)=0.0; product, R_(f)=0.4). Oneobtains 121 g of the important intermediate needed for the preparationof phosphatidyldiglycerols (G₂ parent substance).

[0092] G₂ Parent System:2-0-benzyl-rac-G₁-1.3-0.0-1.2-isopropylidene-rac-G₂ (yield 82%).

[0093] Empirical formula: C₁₆H₂₄O₅ (MW 296.36) calculated: C, 64.85; H,8.16; O, 26.99 measured: C, 64.82; H, 8.14; O, -

[0094] Intermediates which have higher proportions of oligoglycerol andare likewise needed for the production of phosphatidyl oligoglycerolscan be prepared analogously. Some analytical findings pertaining to keyintermediates are summarized below:

[0095] G₃ parent system:2-0-benzyl-rac-G₁-1.3-0.0-2-0-benzyl-rac-G₂-1.3-0.0-1.2-isopropylidene-rac-G₃

[0096] Empirical formula: C₂H₃₆O₇ (MW 460.56) calculated: C, 67,81; H,7.88; O, 24.32 measured: C, 67,75; H, 7.85; O, -

[0097] G₄ Parent System:

[0098]2-0-benzyl-rac-G₁-[1.3-0.0-2-0-benzyl-G]₂-1.3-0.0-1.2-isopropylidene-rac-G₄

[0099] Empirical formula: C₃₆H₄₈O₉ (MW 624.77) calculated: C, 69.21; H,7.74; O, 23.05 measured: C, 69.17; H, 7.69; O,-

[0100] G₆ Parent System:

[0101]2-0-benzyl-rac-G₁-[1.3-0.0-2-0-benzyl-rac-G]₄-1.3-0.0-1.2-isopropylidene-rac-G₆

[0102] Empirical formula: C₅₆H₇₂O₁₃ (MW 953.172) calculated: C, 70.57;H, 7.61; O, 21.82 measured: C, 70.56; H, 7.54; O, -

[0103] G₈ Parent System:

[0104]2-0-benzyl-rac-G₁-[1.3-0.0-2-0-benzyl-rac-G]₆-71.3-0.0-1.2-isopropylidene-rac-G₈

[0105] Empirical formula: C₇₆H₉₆O₁₇ (MW 1281.58) calculated: C, 71.23;H, 7.55; O, 21.22 measured: C, 71,15; H, 7.53; O, -

EXAMPLE 4d:

[0106] Substances which Bear the Tetrahydropyranyl Protective Group(Instead of Benzyl) (Preparation of Phosphatidyl Oligoglycerols whichContain Unsaturated Fatty Acids)

[0107] For this variant, 1.2-isopropylidene-rac-glycero-3-0-allyl etheris prepared and epoxidized as described by H. Eibl and P. Woolley (Chem.Phys. Lipids 41 (1986) 5363).

[0108] Epoxidation (General Example)

[0109] 1.2-isopropylidene-rac-glycero-3-O-allyl ether (MW 172.22; 1mol-172 g) is dissolved in 1 l CH₂Cl₂. 3-chloroperoxybenzoic acid (1.1mol) is added portion-wise and the reaction mixture stirred for 6 hoursat 25-30° C. The educt (R_(f) 0.5 in diethyl ether/pentane 1:1) is bythen transformed completely into the desired product (R_(f) 0.2 in theabove system). After removing the precipitate by suction filtration, 100g Na₂CO₃ is added to the filtrate and the mixture stirred for another 3hours at 20° C. The precipitate is removed and the solvent eliminatedunder vacuum. The yield of epoxide (MW 188.22) is 170 g (90%). Asdescribed under “epoxide opening with alcohols” (Example 4a), theepoxide is now converted with benzyl alcohol into1-0-benzyl-rac-G₁-3.1-0.0-2.3-isopropylidene-rac-G₂ and the free —OHgroups converted with 3.4-dihydro-2H-pyran into the tetrahydropyranderivative.

[0110] Introduction of the Tetrahydropyran Protective Group (GeneralExample)

[0111] 1-0-benzyl-rac-G₁-3.1-0.0-2.3-isopropylidene-rac-G₂ (MW 296.36; 1mol-296 g) is dissolved in 1 l THF, to which 1.4 mol3.4-dihydro-2H-pyran and 0.1 mol toluene sulfonic acid are added. Thereaction is complete after 1 hour (educt, R_(f) 0.65; product, R_(f)0.90 in diethyl ether). 1 l 0.2 mol Na₂CO₃ solution and 1 l diisopropylether are added, and the mixture shaken thoroughly in a separatingfunnel. The upper phase is rotated in an evaporator and the productconverted by means of hydrogenolysis with H₂ in the presence of a PD/Ccatalyst (5% Pd based on the alcohols) into the G₂ constituent with freehydroxyl group.

[0112] G₂ Parent System:2-0-tetrahydropyranyl-rac-G₁-1.3-0.0-1.2-isopropylidene-rac-G₂ (Yield80% Expressed in Terms of the Epoxide)

[0113] Empirical formula: C₁₄H₂₇O₆ (MW 291.36) calculated: C, 57.71; H.9.34; O, 32.95 measured: C, 57.59; H. 9.29; O, -

[0114] Compounds with other parent systems can be converted intoTHP-protected structures in the same way. For example, the 3-0-allylether of example 4a can be converted to an epoxide and opened with allylalcohol. Again, a 3-0-allyl ether is formed, which is epoxidized andopened with benzyl alcohol to form the product below, which, throughintroduction of 3 THP protective groups and catalytic hydrogenolysis,can be converted to an intermediate with the G₄ parent system.

[0115] G₄ Parent System:

[0116]2-O-THP-rac-G₁[1.3-0.O-2-0-THP-rac-G]₂-1.3-0.0-1.2-isopropylidene-rac-G₄

[0117] Empirical formula: C₃₀H₄₅O₂ (MW 607.75) calculated: C, 59.29; H,9.12; O, 31.59 measured: C, 59.24; H, 9.08; O, -

EXAMPLE 4e:

[0118] Further Processing of the Intermediate with the Formula I

[0119] G₂ Parent System (Racemic)

[0120] From formula I, a key intermediate for the preparation of the G₂parent system is obtained (see model B). To this end, the secondary —OHfunction in formula I is alkylated, benzylated, or protected withtetrahydropyran.

[0121] Model B: Key intermediate for preparing the G₂ parent system:

[0122] X=saturated or unsaturated alkyl, benzyl or THP

[0123] Alkyl-G₂ Compounds

[0124] 1) 2-0-alkyl-rac-G₁-1.3-0.0-rac-G₂

[0125] The intermediate compound of formula II in which X=alkyl is freedof the protective groups. The following compounds were isolated:2-O-ethyl-G₂: C₈H₁₈O₅ (194.23) 2-O-hexyl-G₂: C₁₂H₂₆O₅ (250.33)2-O-undecenyl-G₂: C₁₇H₃₄O₅ (318.45) 2-O-dodecyl-G₂: C₁₈H₃₈O₅ (334.49)2-O-octadecyl-G₂: C₂₄H₅₀O₅ (418.65) 2-O-erucyl-G₂: C₂₈H₅₆O₅ (472.75)

[0126] 2) 1-0-alkyl-rac-G₁-3.1-0.0-rac-G₂

[0127] In the intermediate compound of formula If in which X=benzyl,allyl is removed from the 1-position and the corresponding alkyl chainincorporated in the 1-position. Following removal of the protectivegroups, the following compounds were obtained: 1-O-methyl-G₂: C₇H₁₆O₅(180.20) 1-O-propyl-G₂: C₉H₂₀O₅ (208.25) 1-O-nonyl-G₂: C₁₅H₃₂O₅ (292.41)1-O-undecyl-G₂: C₁₇H₃₆O₅ (320.47) 1-O-dodecyl-G₂: C₁₈H₃₈O₅ (334.49)1-O-octadecyl-G₂: C₂₄H₅₀O₅ (418.65)

[0128] Unsaturated 1-O-alkyl diglycerols can also be obtained directlyby way of epoxide opening of 1.2-isopropylidene-glycero-glycidyl ether(Model D, formula IV) with alcohols, eg, 1-O-Undecenyl-G₂: C₁₇H₃₄O₅(318.45)

[0129] However, this path is only suitable for shorter-chain alcohols,since the yields for long-chain alcohols such as oleyl alcohol are low.To prepare 1-oleyl-G₂, therefore, a synthetic pathway via2-O-THP-glycero-1.3.O.O-(1.2-isopropylidene)-glycerol is preferred(model D, formula V)

[0130] 1-O-Oleyl-G₂: C₂₄H₄₈O₅ (416.64)

[0131] Intermediates for the Synthesis of Phospholipids which ContainDiglycerols in the Polar Area

[0132] Compounds with good protective groups for these syntheses containa 2-O-benzyl ether or a 2-O-tetrahydropyranyl ether group in G₁. 1)2-O-benyzl-rac-G₁-1.3-0.0-(1.2-isopropylidene)-rac-G₂: C₁₆H₂₄O₅ (296.36)

[0133] The compound with the formula III is obtained by alkalineallyl/propyl rearrangement, benzylation of the secondary —OH group andsubsequent acidic cleavage of the propenyl protective group.

[0134] Model C: Starting Product for Phosphatidyl Diglycerols withSaturated Fatty Acid Residues. 2)2-O-tetrahydropyranyl-rac-G₁-1.3-0.0-(1.2-isopropylidene)-rac-G₂:C₁₄G₂₇O₆ (291.36)

[0135] The compound with the formula V is made from allyl glycerol. Theintermediate IV is obtained by way of addition of isopropylidenefollowed by epoxidation. After opening the epoxide with benzyl alcohol,the THP protective group is introduced and the benzyl group removed.

[0136] Model D: Starting Product for Phosphatidyl Diglycerols withUnsaturated Fatty Acid Residues G₃ Parent System (Racemic)

[0137] From the key intermediate with the formula If it is possible,with inclusion of the allyl group, to develop triglycerols. Followingepoxidation, various intermediates and end products of pharmaceuticalinterest can be made from the epoxide.

[0138] Model E: Starting Products for Making G₃ Parent Systems

[0139] Triglycerols can be prepared from the key intermediate with theformula VI; the intermediate is also used for making G₄ parent systems.In formula VI, X stands for hydrogen, a saturated alkyl, a benzyl or aTHP residue.

[0140] Alkyl-G₃ Compounds

[0141] 1) 1-O-alkyl-rac-G₁-1.3-0.0-rac-G₂-1.3-0.0-rac-G₃

[0142] The epoxide with the formula V (X=H) is opened directly withalcohols and, after the isopropylidene protective group has been splitoff, results in the following compounds: 1-O-ethyl-G₃: C₁₁H₂₄O₇ (268.30)1-O-hexyl-G₃: C₁₅H₃₂O₇ (324.41) 1-O-nonyl-G₃: C₁₈H₃₈O₇ (366.491)1-O-undecenyl-G₃: C₂₀H₄₀O₇ (392.53) 1-O-dodecyl-G₃: C₂₁H₄₄O₇ (408.57)

[0143] For longer-chain alcohols, direct opening results in poor yields.For this reason, the oleyl and erucyl compounds of G₃ were prepared byopening of V (X=THP) with benzyl, THP-protection of the secondaryhydroxyl groups formed, catalytic debenzylation, alkylation in the 1position and removal of the protective groups. 1-O-oleyl-G₃: C₂₇H₅₄O₇(490.72) 1-O-erucyl-G₃: C₃₁H₆₂O₇ (456.82)

[0144] 2) 2-O-alkyl-rac-G₁-1.3-0.0-rac-G₂-1.3-0.0-rac-G₃

[0145] The epoxide of formula V (X=benzyl or THP) is opened with allylalcohol and alkylated in the 2 position. The protective groups areremoved in the usual way. For the preparation of the unsaturated2-0-alkyl compounds, rearrangement of the allyl protective groups mustprecede alkylation. In addition, only the THP protective group and notbenzyl can be used in G₂ here. The following compounds were prepared:2-O-methyl-G₃: C₁₀H₂₂O₇ (111.99) 2-O-propyl-G₃: C₁₂H₂₆O₇ (282.33)2-O-nonyl-G₃: C₁₈H₃₈O₇ (366.49) 2-O-undecenyl-G₃: C₂₀H₄₀O₇ (392.53)2-O-dodecyl-G₃: C₂₁H₄₄O₇ (408.57) 2-O-hexadecyl-G₃: C₂₅H₅₂O₇ (464.68)2-O-oleyl-G₃: C₂₇H₅₄O₇ (490.72) 2-O-erucyl-G₃: C₃₁H₆₂O₇ (546.82)

[0146] Intermediates for the Synthesis of Phospholipids which ContainTrigylcerides in the Polar Area

[0147] Benzyl and tetrahydropyranyl (THP) residues are convenientprotective groups for synthesizing phospholipids which have G₃ residuesin the polar area. Benzyl residues are readily removed under mildconditions, provided that only saturated fatty acids are used. THPresidues are of particular interest because they can be removed in asingle step together with isopropyl protective groups. 1)2-O-benyzl-rac-G₁-1.3-0.0-(2-O-benzyl)-rac-G₂-1.3-0.0-(1.2-isopropylidene)-rac-G₃ C₂₆H₃₆O₇ (460.56)

[0148] This compound is obtained from the key intermediate VI (X=benzyl)by opening with allyl alcohol, benzylation of the 2 position andcleavage of the allyl protective group. In the text, the compound isreferred to as formula VI. 2)2-O-THP-rac-G₁-1.3-0.0-(2-O-THP)-rac-G₂-1.3-0.0-(1.2-isopropylidene)-rac-G₃: C₂₂H₄₁O₉ (449.56)

[0149] To prepare unsaturated G₃-phospholipids, the residue X=THP isused in VI. The epoxide VI is opened with benzyl alcohol, the secondaryhydroxyl group thus exposed protected with THP, and the benzyl residueremoved catalytically with H₂/Pt. The compound made in this way isreferred to in the text as formula VIII.

[0150] Additional Remarks

[0151] In the description so far we have not made use of the fact thatin formula VI for X=saturated alkyl, compounds of the followingstructure can readily be prepared:1-O-alkyl-rac-G₁-3.1-0.0-(2-0-alkyl)-rac-G₂-3.1-rac-G₃. Therepresentatives of these new structures were made by opening the epoxideVI (X=hexadecyl) with CH₃OH or undecenyl alcohol and splitting off theisopropylidene protective group.1-O-methyl-rac-G₁-3.1-O.O-(2.O-hexadecyl)- rac-G₂-3.1-rac-G₃: C₂₆H₅₄O₇(478.71)  1-O-Undecenyl-rac-G₁-3.1-O.O-(2-O-hexadecyl)-rac-G₂-3.1-O.O-rac-G₃: C₃₆H₇₂O₇ (616.958)

[0152] G₄ Parent System (Racemic)

[0153] G₄ parent systems can be prepared from the key intermediate withthe formula IX.

[0154] Model F: Starting Products for the Preparation of G₄ ParentSystems.

[0155] Tetraglycerols can be made from the key intermediate with theformula IX. They can also be used to prepare pentaglycerols.

[0156] Oligoglycerols with two or more alkyl residues can be made fromthe intermediates, too. Suitable starting compounds here are moleculeswith the formula IX, in which X is a saturated alkyl residue.

[0157] Alkyl-G₄ Compounds

[0158] 1) 1-O-alkyl-rac-G₁-1.3-0.0-rac-G₂-1.3-0.0-rac-G₃-1.3-0.0-rac-G₄

[0159] The epoxide of formula IX, X=H, is opened directly with alcohols.After the isopropylidene protective group has been split off, thefollowing substances are obtained: 1-0-ethyl-G₄: C₁₄H₃₀O₉ (342.38)1-0-hexyl-G₄: C₁₈H₃₈O₉ (398.49) 1-0-undecyl-G₄: C₁₉H₄₀O₉ (412.52)1-0-undecenyl-G₄: C₁₉H₃₈O₉ (410.50) 1-0-dodecyl-G₄: C₂₀H₄₂O₉ (426.54)

[0160] This path is only suitable for shorter-chain alcohols, since theyields are much lower with long-chain alcohols.

[0161] For long-chain, saturated alcohols it is therefore necessary, aswith G₂ and G₃, to select a synthetic pathway via the key intermediatewith X=benzyl. One opens with allyl alcohol, benzylates the thus exposed2-OH group, removes the allyl group in the 1 position and alkylates the1 position. After removing the protective groups one obtains:1-0-hexadecyl-G₄: C₂₄H₅₀O₉ (482.99) 1-0-octadecyl-G₄: C₂₆H₅₄O₉ (510.70)1-0-behenyl-G₄: C₃₀H₆₂O₉ (566.81)

[0162] 2. 2-O-alkyl-rac-G₁-1.3-0.0-rac-G₂-1.3-0.0-rac-G₃-1.2-0.0-rac-G₄

[0163] The key intermediate with the formula IX is opened with allylalcohol, and the thus-exposed 2 position alkylated. After removal of theprotective groups one obtains: 2-0-propyl-G₄: C₅H₃₂O₉ (356.41)2-0-hexyl-G₄: C₁₈H₃₈O₉ (398.49) 2-0-nonyl-G₄: C₂₁H₄₄O₉ (440.57)2-0-undenyl-G₄: C₁₉H₃₈O₉ (410.50) 2-0-dodecyl-G₄: C₂₀H₄₂O₉ (426.54)2-0-hexadecyl-G₄: C₂₄H₄₀O₉ (483.99) 2-0-octadecyl-G₄: C₂₆H₅₄O₉ (510.70)2-0-oleyl-G₄: C₂₆H₅₂O₉ (508.69) 2-0-erucyl-G₄: C₃₀H₆₀O₉ (564.80)

[0164] Intermediates for the Synthesis of Phospholipids which haveTetraglycerols in the Polar Area.

[0165] As with the synthesis of G₂ and G₃ compounds, benzyl andtetrahydropyran ether are suitable protective groups for thesesyntheses. 1)2-O-benzyl-rac-G₁-1.3-0.0-(2-0-benzyl)-rac-G₂-1.3-0.0-(2-0-benzyl)-rac-G₃-1.3-0.0-(1.2-isopropylidene)-rac-G₄: C₃₆H₄₈O₉ (624.77)

[0166] The important intermediate for the synthesis of phospholipidswith G₄ residues in the polar area is made from formula IX, X=benzyl byopening the epoxide with allyl alcohol, benzylating the thus exposed2-OH group and removing allyl. The compound is referred to as formula Xin the text. 2. 2-0-THP-rac-G₁-1.3-0.0-(2-0-THP)-rac-G₂-1.3-0.0-(2-0-THP)-rac-G₃-1.3-0.0-(1.2-isopropylidene)-rac-G₄: C₃₀H₄₅O (607.75)

[0167] To make this compound, which is suitable for obtainingunsaturated phospholipids with G₄ parent systems in the polar area, oneproceeds analogously as for the preparation of the G₃ compound. Oneopens the epoxide VII, X=THP with benzyl alcohol, protects the thusexposed 2-OH group with THP, and removes benzyl with H₂ (Pd/Ccatalysis.). The compound is referred to in the text as formula XI.

[0168] Intermediates for the Synthesis of Phospholipids which ContainOligoglycerols in the Polar Area and Permit an sn-1 Linkage to thePhosphate (Natural Configuration)

[0169] In the preparation of compounds suitable for incorporation in thepolar area of phospholipids (formula III and V for G₂, formula VII andVIII for G₃, formula X and XI for G₄), no attention was paid so far tothe fact that in natural phosphatidylglycerol, ie, in1.2-diacyl-sn-glycero-3-phospho-sn-1-glycerol, the link betweenphosphate and the non-acylated glycerol is an sn-1 linkage. Since theliposome components, as carriers of medicinal products, should be usedin the most natural configuration possible, synthetic pathways weredeveloped which also permit an sn-1 configuration of the polaroligoglycerol (model G).

[0170] sn-1-G₁-G₂ Linkage

[0171] The stereospecific linkage can be obtained using methodsanalogous to those described in the literature (DE 31 30 867 A1; H.Eibl, Chem. Phys. Lipids 28 (1981) 1-5; H. Eibl et al., Chem. Phys.Lipids 41 (1986) 53-63; H. Eibl et al., Chem. Phys. Lipids 47 (1988)47-53).

[0172] The starting product for this linkage is2-O-benzyl-3-O-allyl-sn-glycerol, which, following epoxidation, ishydrolysed to the diol. Following reaction with H⁺/2.2-dimethoxypropane,2-0-Be-sn-G₁-3. 1-0.0-(1.2-isopropylidene)-rac-G₂ is obtained, amolecule with the formula XII, which permits an sn-1 linkage to thephosphate group in phospholipids and corresponds to the racemate offormula III.

[0173] sn-1-G₁-G₂-G₃ Linkage

[0174] The starting product for this linkage is again2-0-benzyl-3-0-allyl-sn-glycerol. Protection of the sn-1 position withTHP is followed by epoxidation, and then the epoxide ring opened with1.2-isopropylidene glycerol. The thus-exposed —OH function in G₂ isbenzylated, and the THP protective group removed. One obtains a moleculewith the formula XIII, which permits an sn-1 linkage to the phosphategroup in phospholipids. The molecule XIII corresponds to the racemate offormula VI.

[0175] sn-1-G₁-G₂-G₃-G₄ Linkage

[0176] The starting product is again 2-0-benzyl-3-0-allyl-sn-glycerol,in order to ensure the sn-1 linkage. Incorporation of the THP protectivegroup is followed by epoxidation, and the epoxide then opened with allylalcohol. Following epoxidation of the intermediate, the epoxide isopened with isopropylidene glycerol, the two exposed —OH groupsbenzylated, and THP removed. One obtains XIV, which permits an sn-1linkage to the phosphate and corresponds to the racemate of formula X.

[0177] If desired, compounds with an sn-3-G₁-G₂, sn-3-G₁-G₂-G₃ orsn-3-G₁-G₂-G₃-G₄ linkage with the phosphate can be made analogously. Inthis case, the same sequence of reactions is required, but instead of2-0-benzyl-2-0-allyl-sn-glycerol, use is made of the enantiomeric2-0-benzyl-1-0-allyl-sn-glycerol.

[0178] Model G: Phospholipid constituents which permit an sn-1-G_(x)linkage (x=2-4). Starting product is 2-O-benzyl-3-O-allyl-sn-glycerol.

EXAMPLE 4f

[0179] Intermediates which Contain Sugar Alcohols (General Examples)

[0180] Important intermediates here are, in particular, such sugaralcohols as are obtainable at a reasonable price or can be made fromthese by means of simple reactions (see enclosed table). Of specialinterest are D-mannitol as open form of inositol, xylitol, which, whenthe middle carbon atom is phophorylated, shows no optical activity andwhich is readily obtained as 1.2; 4.5-diisopropylidene xylitol, andmeso-erythritol. The protective groups chiefly employed here areisopropylidene, trityl in combination with benzyl, or allyl.Tetrahydropyranyl is also of some importance as protective group. Somealternatives will now be described which serve as examples.

[0181] 1.2;4.5-diisopropylidene-xylitol (General Example forIntroduction of the Isopropylidene Protective Group)

[0182] Xylitol (1.0 mol-152 g) is slurried with 500 ml 2-propanol, andmixed with dimethoxypropane (3.0 mol-312 g). Following addition of 6 gH₂SO₄ in 100 ml 2-propanol, the mixture is heated to 50° C. After 30minutes everything has dissolved. Sufficient concentrated ammonia isadded to adjust the reaction mixture to a pH of about 8. The solvent isremoved in a rotary evaporator, and the residue taken up in hexane andcooled to −20° C. White crystals precipitate, which are sucked up andused for the phosphorylation.

[0183] Empirical formula: C₁₁H₁₉O₅ (MW 231.27) calculated: C, 57.13; H,8.28; O, 34.59 measured: C, 57.01; H, 8.27; O, -

[0184] Structural formulae of some sugar alcohols:

[0185] C₄H₁₀O₄ meso-Erythritol D-Threitol L-Threitol

[0186] C₅H₁₂O₅ Adonite (Ribitol) D(+)-Arabitol L(−)-Arabitol Xylitol

[0187] C₆H₁₄O₆ Dulcite (Galactit. D-Mannitol D-Sorbitol

[0188] 1.2;3.4-diisopropylidene-5-benzyl-D-mannitol (General Example forthe use of Trityl Protective Groups Combined with Benzyl ProtectiveGroups)

[0189] Starting with 1.2;3.4;5.6-triisopropylidene-D-mannitol (MW302.36), which is prepared analogously to the xylitol derivative, oneobtains—by means of carefully splitting off the protective group—a1.2.3.4-diisopropylidene-D-mannitol yield of about 30%.Triisopropylidene-D-mannitol (1.0 mol-302 g) is dissolved in 600 mlCH₃OH, to which 15 (5 g) Amberlyst® and 70 g H₂O are then added. Thereaction mixture is heated to 50° C., the solution stirred at thistemperature for 40 minutes (educt, R_(f) 0.9; 1.2;3.4-derivative, R_(f)0.7; 3.4-derivative, R_(f) 0.1 in CHCl₃/CH₃OH 1:1), cooled to 20° C. andfiltered to 7.5 ml 25% ammonia in 25 ml 2-propanol (pH ˜8). Cooling to4° C. causes the starting product to precipitate, which can thus berecovered (ca. 120 g, ˜40%). The filtrate is rotated in an evaporatorand purified chromatographically on silica gel 60 (Merck, Darmstadt).One obtains 84 g (˜32%) of 1.2:3,4-diisopropylidene-D-mannitol, which isretrieved in crystalline form from hexane.

[0190] Empirical formula: C₁₂H₂₂O₆ (MW 262.30) calculated: C, 54.95; H,8.45; O, 36.60 measured: C, 54.89; H, 8.34; O.-

[0191] Reaction of 1.2;3.4-diisopropylidene-D-mannitol with TritylChloride and Benzyl Chloride (General Example for Tritylation andSubsequent Alkylation)

[0192] 1.2:3.4-diisopropylidene-D-mannitol (0.2 mol-52 g) is dissolvedin 300 ml toluene, mixed with triethylamine (0.30 mol-30 g) andreflux-boiled. Trityl chloride (MW 278.78; mol-64 g) in 200 ml tolueneis added dropwise, and the mixture reflux-boiled for another 60 minutes(educt, R_(f) 0.7; product, R_(f) 0.90 in CHCl₃/CH₃OH 10:1). Thereaction is then complete. The mixture is cooled to 20° C., precipitatedtriethylamine hydrochloride filtered off, and the filtrate rotated in anevaporator. The residue is taken up in 400 ml THF, mixed with benzylchloride (0.3 mol-38 g) and reflux-boiled. K-tert. butylate (0.25 mol-28g)—dissolved in 200 ml THF—is added dropwise and the reaction mixtureleft to stand for 1 hour (educt, R_(f) 0.90; product, R_(f) 1.00 inCHCl₃/CH₃OH 10:1). Following addition of 300 ml diisopropyl ether, thereaction mixture is extracted with 600 ml H₂O, the upper phase taken offand the solvent removed under vacuum. The residue can be used directly.

[0193] Cleavage of the Trityl Protective Group while Retaining the3.4-Isopropylidene Protective Group (General Example)

[0194] The oily residue from the preceding reaction (˜0.2 mol) isdissolved in 600 ml ac tone/CH₃OH 1:1, to which 3 ml H₂SO₄ are thenadded. The mixture is stirred at 40° C. for 40 minutes, which results incomplete removal of the trityl- and the 1.2-isopropylidene protectivegroups (educt, R_(f) 0.95; product, R_(f) 0.15 in ether). The reactionmixture is adjusted to pH ˜8, filtered and rotated in an evaporator. Theresidue is purified chromatographically on silica gel, and crystallizedfrom hexane.

[0195] 2-0-benyzl-3.4-isopropylidene-D-mannitol

[0196] Empirical formula: C₁₆H₂₄O₆ (MW 312.36) calculated: C, 61.52; H,7.74; O, 30.73 Measured: C, 61.44; H, 7.72; O, -

[0197] As described for example 4c, the isopropylidene protective groupcan be reintroduced in the 5.6 position. A key intermediate for thesynthesis of phosphatidyl-D-mannitol compounds is obtained, namely2-O-benzyl-3.4;5.6-diisopropylidene-D-mannitol.

[0198] Empirical formula: C₁₉H₂₈O₆ (MW 352.42) calculated: C, 64.75; H,8.01; O, 27.24 measured: C, 64,68; H, 7.94; O, -

[0199] Sugar Alcohol Constituents which are Obtained by SplittingPeriodate off a Vicinal Diol and Reducing the Resulting Aldehyde withSodium Borohydride (General Example)

[0200] 1.2:3.4-diisopropylidene-D-mannitol (0.2 mol-26 g) is dissolvedaccording to the method of H. Eibl (Chem. Phys. Lipids 28 (1981) 1-5) in200 ml CH₃OH and added to a solution of 0.2 mol sodium metaperiodate in500 ml water. The temperature should not exceed 30° C. The reaction iscomplete after 15 minutes. The pH of the reaction mixture is raised topH=8 with 5 M KOH in water. The precipitated salts are filtered off, andthe aldehyde reduced with sodium borohydride (0.25 mol). One obtainsa >90% yield of 1.2:3.4-diisopropylidene-D-lyxitol, which is extractedwith 600 ml of chloroform. The chloroform phase is rotated in anevaporator and the product crystallized from hexane.

[0201] Empirical formula: C₁₁H₁₉O₅ (MW 231.27) calculated: C, 57.13; H,8.28; O, 34.59 measured: C, 57.07; H, 8.21, O, -

[0202] By employing the various alternatives—monoisopropylidenecleavage, periodate cleavage from vicinal diols to produce aldehydeswhich are then reduced with sodium borohydride, and the variationtrityl/alkyl—sugar alcohols are obtained that are protected in verydifferent ways. These can be converted by way of acylation orphosphorylation into interesting alkyl, acyl or phosphatidyl compounds.

[0203] Preparation of Simple Ester and Ether Derivatives from theOligoglycerols and Sugar Alcohols Portrayed (General Description)

[0204] Methods of esterification and etherification, followed bycleavage of the protective groups, have been described in variouspublications. The articles listed below include different methods ofphosphorylation. These methods can be employed here analogously.

[0205] Eibl, H. Synthesis of glycerophospholipids Chem. Phys. Lipids 26(1980) 405-429

[0206] Eibl, H. Phospholipid Synthesis In: Liposomes: From PhysicalStructure to Therapeutic Applications (C. G. Knight, editor) Elsevier,Amsterdam (1981) 19-50

[0207] Eibl, H. and Kovatchev, S. Preparation of phospholipids and oftheir analogues by phospholipase D. In: Methods of Enzymology. Vol. 72.Ed. J. M. Lowenstein, Academic Press, New York (1981) 632-639

[0208] Eibl, H.: Phospholipids als funktionelle Bausteine biologischerMembranen Angew. Chemie 96 (1984) 247-262

[0209] Eibl, H.: Phospholipids as functional constituents ofbiomembranes Angew. Chem. Int. Ed. Engl. 23 (1984) 257-271

[0210] Eibl, H. Phospholipid synthesis: Oxazaphospholanes anddioxaphospholanes as intermediates. Proc. Natl. Acad. Sci. USA 75 (1978)40744077

[0211] Eibl, H. and Wooley, P.: Synthesis of enantiomerically pureglyceryl esters and ethers. I. Methods employing the precursor1,2-isopropylidene-sn-glycerol. Chem. Phys. Lipids 41 (1986) 53-63

[0212] Eibl. H. and Wooley, P.: Synthesis of enantiomerically pureglyceryl esters and ethers. II. Methods employing the precursor3,4-isopropylidene-D-mannitol. Chem. Phys. Kipids 47 (1988) 47-53

[0213] Eibl. H. and Wooley, P.: A general synthetic method forenantiomerically pure ester and ether lysophospholipids. Chem. Phys.Lipids 47 (1988) 63-68

[0214] Wooley, P. and Eibl, H.: Synthesis of enantiomerically purephospholipids including phosphatidylserine and phosphatidylglycerol.Chem. Phys. Lipids 47 (1988) 55-62

Example 4g

[0215] Intermediates for the Synthesis of Phospholipids which ContainSugar Alcohols in the Polar Area

[0216] As already described, the introduction via oligoglycerols ofsubstances in the polar area of phospholipids has a pronounced effect onthe blood circulation if these substances are used as liposomecomponents. The same result can be obtained if, instead of theoligoglycerols, use is made of sugar alcohols, eg, phosphoric acidesters of D-mannitol, D-lyxitol and D-threitol. These compounds can beintroduced with suitable protective groups (see model H) intophospholipids in the manner described for oligoglycerols. With thederivatives described, coupling with phospholipids again leads to ansn-1 linkage between the phosphoric acid and the sugar alcohol.

[0217] D-Mannitol Derivative

[0218] 3.4-0.0-dibenzyl-D-mannitol is prepared from1.2.6.5-diisopropylidene-D-mannitol by benzylating in the 3.4 positionand splitting off the isopropylidene protective groups. Afterintroducing the isopropylidene protective group in the 1.2 position,tritylation and benzylation of the exposed —OH group, one obtains,following cleavage of the trityl group, the compound XV, which can beincorporated in the polar area of phospholipids.

[0219] D-Lyxitol Derivative

[0220] 1.2-isopropylidene-3.4-0.0-dibenyzl-D-mannitol (see above) iscleaved with periodic acid and reduced with NaBH₄ to the alcohol XVI.This compound can be incorporated into the polar area of phospholipids.

[0221] Model H: Polyhydric Alcohols with at Least 4 Hydroxyl Groups forIncorporation into the Polar Area of Phospholipids D-Threitol Derivative

[0222] The compound XVI is converted by way of benzylation into1.2-isopropylidene-3.4.5-0.0.0-tribenyzl-D-lyxitol. After splitting offthe isopropylidene protective group, periodic acid cleavage andreduction to the alcohol, one obtains XVII. This compound can beincorporated into the polar area of phospholipids in the usual way.

[0223] Phospholipids which Contain Oligoglycerols in the Polar Area

[0224] In earlier publications we have described how phospholipids canbe easily prepared from diacyl glycerols with saturated and unsaturatedfatty acid chains, with two identical or two different fatty acid chains(DE 32 39 817 Ar, P. Woolley et al. Chem. Phys. Lipids 47 (1988) 55-62;H. Eibl et al., Chem. Phys. Lipids 47 (1988) 63-68). It is also possibleto use acyl/alkyl or alkyl/acyl glycerols as starting product. However,phospholipids which contain dialkyl glycerols are metabolicallyextremely stable and resorption is negligible.

[0225] Basically, the compounds referred to can be prepared according totwo different methods. This derives from the fact that a phosphoric aciddiester is to be prepared from two alcohols, R₁—OH and R₂—OH.

[0226] The R₁—OH alcohols are alcohols which contain a glycerol backbonewith two fatty acid chains and a free hydroxyl group. They can also havejust one fatty acid chain and an additional protective group, usuallybenzyl, for the preparation of monoacrylic phospholipids; R₁—OH can,however, also stand for an alcohol with a simple alkyl group with one ortwo cis double bonds.

[0227] The R₂—OH alcohols are alcohols which have so far been designatedas G₂, G₃ and G₄ in the text. They are described by the structuralformulae III and V (for G₂), VII and VII (for G₃) and X to XIV (for G₄).In like manner, use can also be made of the sugar alcohol derivatives XVto XVII.

[0228] Model G describes how two alcohols R₁—OH and R₂—OH can beutilized to prepare good yields of phosphoric acid diesters.

[0229] Where, for example, R. =1.2-dipalmitoyl-sn-G and R₂=formula XI,the following structure is obtained after removal of the protectivegroups:

[0230] Model G: Phosphoric Acid Diester with the Formula R₁O—PO⁻ ₃—R₂;Na⁺

[0231] Phosphorus oxychloride is used as phosphorylation agent. From thetwo alcohols R₁OH and R₂OH to be linked via phosphate, one first of allprepares the corresponding phosphoric acid dichloride; this is reactedin each case with the other alcohol to phosphoric acid monochloride.Slightly acid hydrolysis then leads to the phosphoric acid diesters,which, after the protective groups have been split off, form, eg, thesalt XVIII, 1.2-dipalmitoyl-sn-glycero-3-phospho-G₁-G₂-G₃-G₄; Na⁺.

[0232] The following list of examples can be extended at will by usingdifferent combinations of fatty acid chains or by introducing additionalfatty acids, both of synthetic and natural origin. If necessary in orderto obtain particular properties, the phosphatidyl-oligoglycerols cancontain additional alkyl chains or fatty acid residues in theoligoglycerol part.

[0233] The oligoglycerol-based methods described here can thus be usedand modified in manifold ways in order to vary and influence theproperties of liposomes. By analogy with the hexadecylphosphocholinesand the erucylphosphocholines, however, these substances may also beimportant biologically active molecules which influence signaltransduction and thus functional pathways in the cells. Examples ofphospho - G₁-G₂ compounds 1. 1.2-dipalmitoyl-sn-glycero-3-phospho-G₁-G₂;Na⁺ salt: C₄₁H₈₀NaO₁₂P (819.04) 2.1.2-dimyristoyl-sn-glycero-3-phospho-G₁-G₂; Na⁺ salt: C₃₇H₇₂NaO₁₂P(762.93) 3. 1.2-distearoyl-sn-glycero-3-phospho-G₁-G₂; Na⁺ salt:C₄₅H₈₈NaO₁₂P (875.14) 4.1-palmitoyl-2-lauroyl-sn-glycero-3-phospho-G₁-G₂; Na⁺ salt: C₃₇H₇₂NaO₁₂P(762.93) 5. 1-stearoyl-2-lauroyl-sn-glycero-3-phospho-G₁-G₂; Na⁺ salt:C₃₉H₇₆NaO₁₂P (790.98) 6. 1.2-dioleoyl-sn-glycero-3-phospho-G₁-G₂; Na⁺salt: C₄₅H₈₄NaO₁₂P (871.11) 7. 1.2-dierucyl-sn-glycero-3-phospho-G₁-G₂;Na⁺ salt: C₅₃H₁₀₀NaO₁₂P (983.32) 8.1-stearoyl-2-oleoyl-sn-glycero-3-phospho-G₁-G₂; Na⁺ salt: C₄₅H₈₆NaO₁₂P(873.13) 9. 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-G₁-G₂; Na⁺ salt:C₄₃H₈₂NaO₁₂P (845.07) 10.1-stearoyl-2-myristoyl-sn-glycero-3-phospho-G₁-G₂; Na⁺ salt:C₄₁H₈₀NaO₁₂P (819.04) 11.1-stearoyl-2-palmitoyl-sn-glycero-3-phospho-G₁-G₂; Na⁺ salt:C₄₃H₈₄NaO₁₂P (847.09) 12. 1-myristoyl-sn-glycero-3-phospho-G₁-G₂; Na⁺salt: C₂₃H₄₆NaO₁₁P (552.57) 13. 1-palmitoyl-sn-glycero-3-phospho-G₁-G₂;Na⁺ salt: C₂₅H₅₀NaO₁₁P (580.62) 14.1-stearoyl-sn-glycero-3-phospho-G₁-G₂; Na⁺ salt: C₂₇H₅₄NaO₁₁P (608.68)15. Erucyl-phospho-G₁-G₂; Na⁺ salt: C₂₈H₅₆NaO₈P (574.71) 16.Octadecyl-phospho-G₁-G₂; Na⁺ salt: C₂₄H₅₀NaO₈P (520.62) 17.Hexadecyl-phospho-G₁-G₂; Na⁺ salt: C₂₂H₄₆NaO₈P (492.56) 18.Tetradecyl-phospho-G₁-G₂; Na⁺ salt: C₂₀H₄₂NaO₈P (464.51) 19.Oleyl-phospho-G₁-G₂; Na⁺ salt: C₂₄H₄₈NaO₈P (518.60) 20.1-O-octadecyl-2-O-methyl-sn-glycero-3-phospho-G₁-G₂; Na⁺ salt:C₂₈H₅₈NaO₁₀P (608.72) Examples of phospho-G₁-G₂-G₃ compounds 1.1.2-dipalmitoyl-sn-glycero-3-phospho-G₁-G₂-G₃; Na⁺ salt: C₄₄H₈₆NaO₁₄P(893.12) 2. 1.2-distearoyl-sn-glycero-3-phospho-G₁-G₂-G₃; Na⁺ salt:C₄₈H₉₄NaO₁₄P (949.22) 3.1-palmitoyl-2-lauroyl-sn-glycero-3-phospho-G₁-G₂-G₃; Na⁺ salt:C₄₀H₇₈NaO₁₄P (837.01) 4.1-stearoyl-2-lauroyl-sn-glycero-3-phospho-G₁-G₂-G₃; Na⁺ salt:C₄₂H₈₂NaO₁₄P (865.06) 5. 1.2-dioleoyl-sn-glycero-3-phospho-G₁-G₂-G₃; Na⁺salt: C₄₈H₉₀NaO₁₄P (945.19) 6.1.2-dierucyl-sn-glycero-3-phospho-G₁-G₂-G₃; Na⁺ salt: C₅₆H₁₀₆NaO₁₄P(1057.40) 7. 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-G₁-G₂-G₃; Na⁺salt: C₄₈H₉₂NaO₁₄P (947.21) 8.1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-G₁-G₂-G₃; Na⁺ salt:C₄₆H₈₈NaO₁₄P (919.148) 9. 1-stearoyl-sn-glycero-3-phospho-G₁-G₂-G₃; Na⁺salt: C₃₀H₆₀NaO₁₃P (682.76) 10. Erucyl-phospho-G₁-G₂-G₃; Na⁺ salt:C₃₁H₆₂NaO₁₀P (648.79) 11. Octadecyl-phospho-G₁-G₂-G₃; Na⁺ salt:C₂₇H₅₆NaO₁₀P (594.69) 12. Hexadecyl-phospho-G₁-G₂-G₃; Na⁺ salt:C₂₅H₅₂NaO₁₀P (566.64) 13.3-O-octadecyl-2-O-methyl-sn-glycero-1-phospho-G₁-G₂-G₃; Na⁺ salt:C₃₁H₆₄NaO₁₂P (682.80) Examples of phospho-G₁-G₂-G₃-G₄ compounds 1.1.2-dipalmitoyl-sn-glycero-3-phospho-G₁-G₂-G₃-G₄; Na⁺ salt: C₄₇H₉₂NaO₁₆P(967.20) 2. 1.2-distearoyl-sn-glycero-3-phospho-G₁-G₂-G₃-G₄; Na⁺ salt:C₅₁H₁₀₀NaO₁₆P (1023.30) 3.1-stearoyl-2-lauroyl-sn-glycero-3-phospho-G₁-G₂-G₃-G₄; Na⁺ salt:C₄₅H₈₈NaO₁₆P (939.14) 4. 1.2-dioleoyl-sn-glycero-3-phospho-G₁-G₂-G₃-G₄;Na⁺ salt: C₅₁H₉₆NaO₁₆P (1019.27) 5.1.2-dierucyl-sn-glycero-3-phospho-G₁-G₂-G₃-G₄; Na⁺ salt: C₅₉H₁₁₂NaO₁₆P(1131.48) 6. 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-G₁-G₂-G₃-G₄; Na⁺salt: C₅₁H₉₈NaO₁₆P (1021.29) 7. Erucyl-phospho-G₁-G₂-G₃-G₄; Na⁺ salt:C₃₄H₆₈NaO₁₂P (722.87) Examples of phospho-sn-G₁ compounds sn-1-G₁-G₂: 1.1.2-dipalmitoyl-sn-glycero-3-phospho-sn-1-G₁-G₂; Na⁺ salt: C₄₁H₈₀NaO₁₂P(819.04) 2. 1.2-distearoyl-sn-glycero-3-phospho-sn-1-G₁-G₂; Na⁺ salt:C₄₅H₈₈NaO₁₂P (875.14) 3.1-stearoyl-2-lauroyl-sn-glycero-3-phospho-sn-1-G₁-G₂; Na⁺ salt:C₃₉H₇₆NaO₁₂P (790.98) 4.1-stearoyl-2-oleoyl-sn-glycero-3-phospho-sn-1-G₁-G₂; Na⁺ salt:C₄₅H₈₆NaO₁₂P (873.13) sn-1-G₁-G₂-G₃: 1.Dipalmitoyl-sn-glycero-3-phospho-sn-1-G₁-G₂-G₃; Na⁺ salt: C₄₄H₈₆NaO₁₄P(893.12) 2. 1.2-distearoyl-sn-glycero-3-phospho-sn-1-G₁-G₂-G₃; Na⁺ salt:C₄₈H₉₄NaO₁₄P (949.22) sn-1-G₁-G₂-G₃-G₄: 1.1.2-dipalmitoyl-sn-glycero-3-phospho-sn-1-G₁-G₂-G₃-G₄; Na⁺ salt:C₄₇H₉₂NaO₁₆P (967.20) 2.1.2-distearoyl-sn-glycero-3-phospho-sn-1-G₁-G₂-G₃-G₄; Na⁺ salt:C₅₁H₁₀₀NaO₁₆P (1023.30) Examples of linkages with sugar alcoholsPhospho-D-mannitol compounds 1.1.2-dipalmitoyl-sn-glycero-3-phospho-D-mannitol; Na⁺ salt: C₄₁H₈₀NaO₁₃P(835.03) 2. 1.2-distearoyl-sn-glycero-3-phospho-D-mannitol; Na⁺ salt:C₄₅H₈₈NaO₁₃P (891.13) 3.1-palmitoyl-2-lauroyl-sn-glycero-3-phospho-D-mannitol; Na⁺ salt:C₃₇H₇₂NaO₁₃P (788.92) 4.1-stearoyl-2-lauroyl-sn-glycero-3-phospho-D-mannitol; Na⁺ salt:C₃₉H₇₆NaO₁₃P (806.97) 5.1-stearoyl-2-myristoyl-sn-glycero-3-phospho-D-mannitol; Na⁺ salt:C₄₁H₈₀NaO₁₃P (835.03) 6. 1-stearoyl-sn-glycero-3-phospho-D-mannitol; Na⁺salt: C₂₇H₅₄NaO₁₂P (624.67) 7. Octadecyl-phospho-D-mannitol; Na⁺ salt:C₂₄H₅₀NaO₉P (536.61) 8.1-O-octadecyl-2-O-methyl-sn-glycero-3-phospho-D-mannitol; Na⁺ salt:C₂₈H₅₈NaO₁₁P (624.71) Phospho-D-lyxitol compounds 1.1.2-dipalmitoyl-sn-glycero-3-phospho-D-lyxitol; Na⁺ salt: C₄₀H₇₈NaO₁₂P(805.00) 2. 1.2-distearoyl-sn-glycero-3-phospho-D-lyxitol; Na⁺ salt:C₄₄H₈₆NaO₁₂P (861.10) 3.1-palmitoyl-2-lauroyl-sn-glycero-3-phospho-D-lyxitol; Na⁺ salt:C₃₆H₇₀NaO₁₂P (758.89) 4.1-stearoyl-2-lauroyl-sn-glycero-3-phospho-D-lyxitol; Na⁺ salt:C₃₈H₇₄NaO₁₂P (776.94) 5.1-stearoyl-2-myristoyl-sn-glycero-3-phospho-D-lyxitol; Na⁺ salt:C₄₀H₇₈NaO₁₂P (805.00) Phospho-D-threitol compounds 1.1.2-dipalmitoyl-sn-glycero-3-phospho-D-threitol; Na⁺ salt: C₃₉H₇₆NaO₁₁P(774.97) 2. 1.2-distearoyl-sn-glycero-3-phospho-D-threitol; Na⁺ salt:C₄₃H₈₄NaO₁₁P (831.07) 3.1-stearoyl-2-lauroyl-sn-glycero-3-phospho-D-threitol; Na⁺ salt:C₃₇H₇₂NaO₁₁P (746.91) 4.1-stearoyl-2-myristoyl-sn-glycero-3-phospho-D-threitol; Na⁺ salt:C₃₉H₇₆NaO₁₁P (774.97)

Example 4h

[0234] Phosphorylation Steps (General Directions) Based, by way ofExample, on the Isolation of1.2-dipalmitoyl-sn-glycero-3-phospho-glyceroglycerol, Na⁺ Salt

[0235] POCl₃ (0.1 mol-15.3 g) in 15 ml THF is introduced into athree-necked flask. While vigorously stirring the contents of theice-cooled flask, one adds—dropwise—1.2-dipalmitoyl-sn-glycerol (0.1mol-57 g) in 100 ml THF and, separately, triethylamine (0.11 mol-11 g)in such manner that there is always a slight excess of triethylaminecompared to 1.2-dipalmitoyl-sn-glycerol, which takes up the HCl as itforms. The temperature of the reaction mixture should not exceed 16° C.On completion of the addition, the reaction mixture is left to stand fora further 30 minutes at 16° C. and then subjected to a TLC check to makesure that the reaction is complete (1.2-dipalmitoyl-sn-glycerol, R_(f)0.8; 1.2-dipalmitoyl-sn-glycero-3-phosphoric acid dichloride isconverted by way of methanolysis to the corresponding phosphoric aciddimethyl ester, R_(f) 0.4 in ether.)

[0236] The second phosphorylation step is carried out with a protectedoligoglycerol. Here, the conversion with2-O-benzyl-rac-G₁-1.3-0.0-1.2-isopropylidene-rac-G₂ is described. To thereaction mixture with 1.2-dipalmitoyl-sn-glycero-3-phosphoric aciddichloride one adds—dropwise—the above alcohol (0.105 mol-31 g) andtriethylamine (0.13-13 g) in 100 ml THF in such manner that thetemperature of the reaction mixture does not exceed 40° C. After 3 hoursat 40° C. the reaction is complete (starting product phosphoric aciddimethyl ester, R_(f) 0.4; product methyl ester, R_(f) 0.7 in ether).One removes the trethylamine hydrochloride precipitate by filtration andhydrolyses the reaction mixture, mainly1.2-dipalmitoyl-sn-glycero-3-phospho-2-O-benzyl-rac-glycero-1.3-0.0-1.2-isopropylidene-rac-glycerol-monochloridetogether with incompletely reacted1.2-dipalmitoyl-sn-glycero-3-phosphoric acid dichloride, with 26 gNa₂CO₃ dissolved in 260 ml H₂O. After 4 hours, 400 ml diisopropyl etherare added and the upper phase, which contains the product, rotated in anevaporator until crystals begin to form. 500 ml acetone are now added,and the crystals formed removed under suction at 20° C. The filtratecontains the protected phosphatidylglyceroglycerol, Na⁺ salt (R_(f) 0.6in CHCl₃/CH₃OH/glacial acetic acid/H₂O 600:60:20:5). After removal ofthe solvent, one obtains 48 g crude product, which is heated in 140 mlacetic acid and 60 ml H₂O for 30 minutes to 60-70° C. (cleavage of theisopropylidene protective group). One then adds 500 ml CHCl₃, 600 mlCH₃OH and 400 ml H₂O and shakes thoroughly. The lower CHCl₃ phase iswashed again with 600 ml CH₃OH and 500 ml H₂O, with addition ofsufficient Na₂CO₃ to obtain a pH of 6 in the aqueous phase. The lowerchloroform phase is rotated in an evaporator and the residue taken up in400 ml THF. To remove the benzyl protective group, the solution has 6 gPd/C added to it and is de-benzylated in a H₂ atmosphere. The reactionis complete after about 4 hours. The catalyst is separated off byfiltration, the solvent removed and the residue (−30 g) taken up in 100ml CHCl₃. 900 ml acetone are added, and the crystals formed removedunder suction. One obtains a white powder;1.2-dipalmitoyl-sn-glycero-3-phosphoglyceroglycerol, Na⁺ salt, yield: 26g (−32%).

[0237] Empirical formula: C₄, H₈₀NaO₁₂P (MW 819.04) calculated: C,60.13; H, 9.85; Na, 2.81; 0, 23.44; P, 3.78 measured: C, 60.01; H, 9.79;Na, -; 0, -; P, 3.69

[0238] 1.2-dipalmitoyl-sn-glycero-3-phospho-glycero-glycero-glycero, Na⁺Salt

[0239] Empirical formula: C₄₄H₈₆NaO₁₄P (MW 893.12) calculated: C, 59.17;H, 9.71; Na, 2.57; 0, 25.08; P, 3.47 measured: C, 59.11; H, 9.62; Na, -;0, -; P, 3.45

[0240] 1.2-dipalmitoyl-sn-glycero-3-phospho-glycero-glycero-glycerol,Na⁺ Salt

[0241] Empirical formula: C₄₇H₉₂NaO₁₆P (MW 967.20) calculated: C, 58.37;H, 9.59; Na, 2.38; O, 26.47; P, 3.20 measured: C, 58.29; H, 9.53; Na, -;O, -; P, 3.19

[0242] To prepare phosphatidyl-oligoglycerols with an aliphatic chain,the so-called lysophosphatidyloligoglycerols, one can start withcompounds which have a benzyl ether group in the sn-2 position of theglycerol, eg, 1-palmitoyl-2-O-benzyl-sn-glycerol,1-stearoyl-2-O-benzyl-sn-glycerol, 1-O-hexadecyl-2-O-benzyl-sn-glycerol,1-O-octadecyl-2-O-benzyl-sn-glycerol etc. We have described thepreparation of these compounds in the publications H. Eibl and P.Woolley, Chem. Phys. Lipids 41 (1986) 53-63 and Chem. Phys. Lipids 47(1988) 55-62. They are phosphorylated in the manner described for thepreparation of 1.2-dipalmitoyl-sn-glycero-3-phospho-glyceroglycerol, Na⁺salt, and reacted with the protected oligoglycerols. The protectivegroups are split off analogously. In the last step, by means ofcatalytic hydrogenolysis with Pd/C (5% on activated charcoal) the benzylgroups are split off both the oligoglycerol part and the glycerol, whichcarries an acyl or alkyl group (see the examples).

[0243] Preparation of the alkylphospho-oligoglycerols is easy bycomparison, as in this case the corresponding alcohols are reactedaccording to the given phosphorylation pattern. To obtain theunsaturated sorts, however, one must use tetrahydropyranyl instead ofbenzyl as protective group.

[0244] A different strategy altogether is employed to synthesize theunsaturated representatives of this substance group.1.2-dibenzyl-sn-glycerol is phosphorylated in the described manner (see,in addition, German patent application DE 32 39 817), then reacted witha tetrahydropyranyl-protected oligoglycerol. Instead of the hydrolysis,a methanolysis is performed and phosphoric acid triesters obtained, eg,for 2-O-tetrahydropyranyl-rac-G₁-1.3-0.0-1.2-isopropylidene-rac-G₂;

[0245] This key intermediate is now subjected to hydrolysis with Pd/C(5% on activated charcoal). One obtains:

[0246] It is now possible to introduce arbitrary unsaturated andsaturated fatty acids at the sn-1 and sn-2 positions of the glycerolmolecule. This step is followed, as described in our earlier patentapplication, by splitting off the methyl group with LiBr and thenhydrolysing the isopropylidene and tetrahydropyranyl protective groupsin 70% acetic acid at 60 to 70° C. The dioleoyl compounds do notcrystallize readily and must therefore be purified chromatographically;the dierucyl compounds, by contrast, are obtained easily in cristallineform.

[0247] The phosphoric acid triester strategy is also recommended forpreparing mixed-chain phosphatidyl-oligoglycerols. In this case, as withthe synthesis of lysophosphatidyloligoglycerols,1-acyl-2-O-benzyl-sn-glycerols or 1-O-alkyl-2-O-benzyl-sn-glycerols areused as starting products and reacted analogously to1.2-dibenzyl-sn-glycerol. Following catalytic debenzylation one obtainsas intermediates for the G₂ compound:

[0248] Unsaturated fatty acids can now be introduced at the free sn-2position, and the molecule freed from its protective groups asdescribed. It is convenient that molecules with the fatty acidcombination 1-palmitoyl-2-oleoyl- or 1-stearoyl-2-oleoyl crystallizereadily.

EXAMPLE 5 Additional Properties

[0249] As the number of glycerol molecules increases, the number of freehydroxyl groups and thus also the polarity increases. For a proportionof 5% in water, PP-G-PG₄ is the only PP-G-PG_(n) to form a clearisotropic solution. The lipids PP-G-PG₁, PP-G-PG₂ and PP-G-PG₃ dissolvewhen heated above 40° C. in water and form superstructures. When thetemperature falls below 40° C., lamellar structures with differentlypronounced hystereses are formed, which are recognizable on account oftheir anisotropy (birefraction in polarized light). The PP-G-PG₁solution becomes cloudy most quickly, and excess lipid precipitates whenthe solution is left to stand at room temperature. The lamellar phasesof PP-G-PG₂ and PP-G-PG₃ remain stable even at low temperatures (down to4° C.). Whereas the transition from the isotropic to the anisotropicphase at room temperature is recognizable after a few minutes withPP-G-PG₂, it takes several hours for PP-G-PG₃. The differences inpolarity are also evident from the different retention factors (R_(f))in the thin-layer chromatogram on silica gel.

What is claimed is:
 1. A compound of the formula (A):

wherein: R₁ and R₂ are independently hydrogen or a saturated orunsaturated C₈-C₂₄ alkyl or C₈-C₂₄ acyl residue, which may be branchedor substituted, x is a whole number from 1 to 4; and m is a whole numberfrom 2 to 5, wherein the compound is more than 90% uniform with respectto the value of m.
 2. The compound of claim 1, wherein the compound ismore than 95% uniform with respect to the value of m.
 3. The compound ofclaim 1, wherein the compound is more than 99% uniform with respect tothe value of m.
 4. The compound of claim 1, wherein x=1 and m is a wholenumber from 2 to
 4. 5. The compound of claim 2, wherein x=1 and m is awhole number from 2 to
 4. 6. The compound of claim 1, wherein at leastone of the residues R₁ and R₂ is an acyl residue.
 7. The compound ofclaim 1, wherein only one of R₁ and R₂ is [CH₃(CH₂)₁₆CO].
 8. Thecompound of claim 1, wherein both R₁ and R₂ are [CH₃(CH₂)₁₆CO].
 9. Thecompound of claim 8, wherein x=1 and m=2.
 10. The compound of claim 8,wherein x=1 and m=3.
 11. A compound represented by the formula:


12. A compound represented by the formula:


13. A liposome comprising a phospholipid, alkyl phospholipid, orcholesterol, and 1 to 50 mol % of a compound of the formula (A):

or salts thereof; R₁ and R₂ are independently hydrogen or a saturated orunsaturated C₈-C₂₄ alkyl or C₁-C₂₄ acyl, which may be branched orsubstituted; x is a whole number from 1 to 4; and m is a whole numberfrom 2 to 5; wherein the compound of formula (A) is more than 90%uniform with respect to the value of m, provided that when the liposomecontains a phospholipid, the phospholipid and the compound of formula(A) contain the same fatty acid residues.
 14. The liposome of claim 13,wherein the compound of formula (A) is more than 95% uniform withrespect to the value of m.
 15. The liposome of claim 13, wherein thecompound of formula (A) is more than 99% uniform with respect to thevalue of m.
 16. The liposome of claim 13, wherein x=1 and m is a wholenumber from 2 to
 4. 17. The liposome of claim 14, wherein x=1 and m is awhole number from 2 to
 4. 18. The liposome of claim 13, wherein at leastone of the residues R₁ and R₂ is an acyl residue.
 19. The liposome ofclaim 13, wherein only one of R₁ and R₂ is [CH₃(CH₂)₁₆CO].
 20. Theliposome of claim 13, wherein both R₁ and R₂ are [CH₃(CH₂)₁₆CO].
 21. Theliposome of claim 20, wherein x=1 and m=2.
 22. The liposome of claim 20,wherein x=1 and m=3.
 23. The liposome of claim 13, wherein the compoundof formula (A) is:


24. The liposome of claim 13, wherein the compound of formula (A) is:


25. The liposome of claim 13, wherein the liposome contains 5 to 15 mol% of the compound of formula (A).
 26. A pharmaceutical compositioncomprising a liposome and a pharmaceutical drug, said liposomecomprising a phospholipid, alkyl phospholipid, or cholesterol, and 1 to50 mol % of a compound of formula (A):

or salts thereof; R₁ and R₂ are independently hydrogen or a saturated orunsaturated C₈-C₂₄ alkyl or C₈-C₂₄ acyl residue, which may be branchedor substituted; x is a whole number from 1 to 4; and m is a whole numberfrom 2 to 5; wherein the compound of formula (A) is more than 90%uniform with respect to the value of m, provided that when the liposomecontains a phospholipid, the phospholipid and the compound of formula(A) contain the same fatty acid residues, and wherein the pharmaceuticaldrug is entrapped within said liposome.
 27. The pharmaceuticalcomposition of claim 26, wherein said composition is combined with apharmaceutical diluent, adjuvant, carrier medium, or filler.
 28. Thepharmaceutical composition of claim 26, wherein said drug is acytostatic agent.
 29. The pharmaceutical composition of claim 28,wherein said cytostatic agent is an anthracycline antibiotic.
 30. Thepharmaceutical composition of claim 29, wherein said anthracyclineantibiotic is a member selected from the group consisting ofdoxorubicin, epirubicin, and daunomycin.
 31. The pharmaceuticalcomposition of claim 26, wherein said drug is an anti-tumor drug. 32.The pharmaceutical composition of claim 31, wherein said anti-tumor drugis a member selected from the group consisting of idarubicin,hexadecylphosphocholine,1-octadecyl-2-methyl-rac-glycero-3-phosphocholine, a cis-platinumcomplex, and a mitomycin.
 33. The pharmaceutical composition of claim32, wherein the cis-platinum complex is a member selected from the groupconsisting of carboplatin and novantron.
 34. The pharmaceuticalcomposition of claim 26, wherein said drug is an immunomodulatingsubstance.
 35. The pharmaceutical composition of claim 34, wherein theimmunomodulating substance is a cytokine.
 36. The pharmaceuticalcomposition of claim 35, wherein said cytokine is an interferon.
 37. Thepharmaceutical composition of claim 36, wherein the interferon isα-interferon.
 38. The pharmaceutical composition of claim 26, whereinsaid drug is an antimycotic substance.
 39. The pharmaceuticalcomposition of claim 38, wherein said antimycotic is amphotericin B. 40.The pharmaceutical composition of claim 26, wherein said drug is ananti-protozoal drug.
 41. The pharmaceutical composition of claim 26wherein said drug is Taxol.
 42. The pharmaceutical composition of claim26, wherein said drug is a lytic drug.
 43. The pharmaceuticalcomposition of claim 42, wherein said lytic drug is a member selectedfrom the group consisting of miltefosin, edelfosin, ilmofosin, andSR162-834.
 44. The pharmaceutical composition of claim 26, wherein thecompound of formula (A) is:


45. The pharmaceutical composition of claim 26, wherein the compound offormula (A) is:


46. A method of prolonging the time a pharmaceutical drug circulates inthe blood of a subject, the method comprising: introducing into thesubject's bloodstream a pharmaceutical composition comprising a liposomeand the pharmaceutical drug, said liposome comprising a phospholipid,alkyl phospholipid, or cholesterol, and 1 to 50 mol % of a compound offormula (A):

or salts thereof; R₁ and R₂ are independently hydrogen or a saturated orunsaturated C₈-C₂₄ alkyl or C₈-C₂₄ acyl residue, which may be branchedor substituted; x is a whole number from 1 to 4; and m is a whole numberfrom 2 to 5; wherein the compound of formula (A) is more than 90%uniform with respect to the value of m, provided that when the liposomecontains a phospholipid, the phospholipid and the compound of formula(A) contain the same fatty acid residues, and wherein the pharmaceuticaldrug is entrapped within said liposome.
 47. The method of claim 46,wherein the compound of formula (A) is more than 95% uniform withrespect to the value of m.
 48. The method of claim 46, wherein thecompound of formula (A) is more than 99% uniform with respect to thevalue of m.
 49. The method of claim 46, wherein x=1 and m is a wholenumber from 2 to
 4. 50. The method of claim 47, wherein x=1 and m is awhole number from 2 to
 4. 51. The method of claim 46, wherein at leastone of the residues R₁ and R₂ is an acyl residue.
 52. The method ofclaim 46, wherein only one of R₁ and R₂ is [CH₃(CH₂)₁₆CO].
 53. Themethod of claim 46, wherein both R₁ and R₂ are [CH₃(CH₂)₁₆CO].
 54. Themethod of claim 53, wherein x=1 and m=2.
 55. The method of claim 53,wherein x=1 and m=3.
 56. The method of claim 46, wherein the compound offormula (A) is:


57. The method of claim 46 wherein the compound of formula (A) is: